Project Description This proposal aims to linearize, immobilize and perform super-resolution imaging of native chromatin fibers using a platform of elastomeric nanochannels. The immediate biological goal is to track the transmission of histones during DNA replication and the redistribution of histone modifications during transcription activation, revealing how epigenetic information is coordinated on individual chromatin fibers. Conventional nanochannels, while capable of linearizing DNA and chromatin, do not allow super-resolution imaging due to mobility of the biopolymers within the channels. This proposal will overcome this key limitation by using completely collapsible elastomeric nanochannels to not only linearize chromatin but also immobilize it. The required nanochannels will be fabricated by guided fracture of elastomer-sandwiched brittle thin films. The ?tunable? channels are opened to enable efficient loading of the relatively large chromatin in their coiled states. Then the channels are gradually closed in several steps. The combined hydrodynamic flow and confinement within the nanochannel linearize and immobilize individual chromatin fibers, which will be imaged and mapped with super-resolution optical microscopy. The process can be reversed and repeated, allowing the same chromatin fiber to be examined multiple times. This will not only improve confidence of the chromatin status revealed, but also enable us to study various epigenetic marks, particularly histone modifications, on the same chromatin fiber. For biology, we will focus on Tetrahymena rDNA mini-chromosome, which can also be engineered as a high copy number expression vector. Its size (~20 kb) is optimal for linearization in nanochannels. Labelling with fluorescent proteins or antibodies coupled with fluorescent dyes enables optical differentiation of old and new histones, as well as various histone modifications. This will allow direct visualization of how histones are transmitted during DNA replication. We will also examine redistribution of various histone modifications accompanying transcription activation. Importantly, we will be able to directly examine the status of histones and histone modifications in nucleosomes discretely positioned in individual chromatin fibers. This will reveal the connectivity or coordination between different epigenetic marks. The aims of this project are: Aim 1. Computational analysis of fabrication and operation of normally-closed tunable nanochannels Aim 2. Construction of straining system, and optimization of chromatin linearization and imaging Aim 3. Analysis of histones and histone modifications on individual chromatin fibers We will examine the inheritance of old histones during DNA replication, in normal as well as replicative stress conditions. We will also analyse the redistribution of histone variants and histone modifications, upon induction of transcription activation or silencing. These studies will be extended to mutants deficient in epigenetic pathways, as well as in pharmacologically perturbed cells.